Therapeutic treatment by intramammary infusion

ABSTRACT

A method of treating, by intramammary infusion of liposomes, Brucella spp. infections in an animal by administration of a therapeutically effective amount of aminoglycoside in liposome form, also being a method of administering a therapeutic agent in liposome form to a proximal mammary lymph node or mammary tissue of an animal.

The present invention is a continuation-in-part of copending U.S. patentapplication Ser. No. 946,398, filed Dec. 23, 1986, and copending U.S.patent application Ser. No. 660,573 filed Oct. 12, 1984 which is in turna continuation-in-part of U.S. patent application Ser. No. 476,496 filedMar. 24, 1983 and now U.S. Pat. No. 4,522,803 to Lenk et. al issued June11, 1985.

FIELD OF THE INVENTION

The present invention concerns a method of treating, by intramammaryinfusion of liposomes, Brucella spp. infections in an animal byadministration of a therapeutically effective amount of aminoglycosidein liposome form, also being a method of administering a therapeuticagent to a proximal mammary lymph node or mammary tissue of an animal.

BACKGROUND OF THE INVENTION

Many workers have attempted to develop an effective and practicalchemotherapeutic regimen for brucellosis in animals, including humans,and particularly in dairy animals such as camels, cows, sheep and goats.The goal has been to salvage farm animals with superior production andbreeding potential. Furthermore in many countries the slaughter ofinfected animals is not possible for financial reasons and healthyreplacements may not be available. Clearly, a short term treatmentregimen would be of major benefit to animal husbandrymen world wide.Furthermore, infected animals are a vector for the infection of humansusing contaminated dairy products.

It is known that in vitro intraphagocytic killing of Brucella abortus inbovine mononuclear leukocytes was enhanced by multilamellar liposomescontaining the aminoglycoside gentamicin. Dees, C., et al., "EnhancedIntraphagocytic Killing of Brucella abortus in Bovine Mononuclear Cellsby Liposomes Containing Gentamicin", Vet. Immunol. and Immunopath,8:171-182 (1985). In vitro killing of Brucella abortus was also enhancedwhen compared to free gentamicin. Other studies found that stableplurilamellar vesicle-entrapped aminoglycosides administered to Brucellacanis infected mice and Brucella abortus infected guinea pigseffectively eliminated bacteria from the organs. Fountain, M. W., et al,"Treatment of Brucella canis and Brucella abortus in Vitro and in Vivoby Stable plurilamellar Vesicle-Encapsulated Aminoglycosides", J. Inf.Dis., 152:529-535 (1985).

Recent studies found that a combination of a long-acting tetracyclinesuch as oxytetracycline (e.g., LA-200™, Pfizer, Terra haute, IN) andstreptomycin apparently cured 67% of infected dairy cows. Milward, F.W., "Effectiveness of Various Therapeutic Regimens for BovineBrucellosis", Am. J. Vet. Res., 45:1825-88 (1984); Nicoletti, P. W.,"Efficacy of Long-Acting Oxytetracycline Alone or Combined withStreptomycin in the treatment of Brucellosis", Vet. Med. Assoc,187:493-95 (1986). Other studies indicated that extension of treatmentimproved the cure rate to over 90%. However the treatment regimenrequired was both lengthy and laborious, requiring approximately onemonth maintainence of therapeutically effective levels of antibiotic intissue. Therefore, attempts have been made to reduce the length anddifficulty of treatment and increase the efficiency of the antibioticsemployed.

Treatment of pathological mammary conditions has been beset with theproblem of administering therapeutically effective levels of therapeuticagent into the mammary tissue with special reference to the lymphaticsystem serving the mammary gland, the proximal mammary lymph nodes.Infections and neoplasms of mammary tissue and proximal lymph nodes haveproved resistant to treatment.

SUMMARY OF THE INVENTION

The instant invention presents a method of treating Brucella spp.infections in an animal, including humans, and particularly dairyanimals, by administration of a therapeutically effective amount ofaminoglycoside in liposome form by intramammary infusion. In a preferredembodiment the aminoglycoside is streptomycin, which is administered ata dosage of about 35 to about 50 mg/kg body weight.

An effective dosage regimen comprises administering the streptomycin inabout 2 to about 5 doses.

The aminoglycoside is preferably administrable in conjunction with atherapeutically effective amount of a tetracycline, includinglong-acting oxytetracycline (e.g., oxytetracycline in a pyrrolidinecarrier), particularly, as to oxytetracycline, at from about 10 to about30 mg/kg body weight.

The liposome form preferred in this invention is stable plurilamellarvesicles, which in one embodiment comprise phosphatidylcholine.

In a particular embodiment the liposomes comprise at least about equalamounts of lipid and streptomycin sulfate (w/w).

This invention further includes a method of administering a therapeuticagent to a proximal mammary lymph node of an animal comprisingintramammary infusion of the therapeutic agent in liposome form.

In various embodiments the therapeutic agent is an anti-infective agentor antineoplastic agent, and/or the liposomes are stable plurilamellarvesicles, and/or the liposomes comprise phosphatidylcholine.

Preferred anti-infective agents are imidazoles and β-lactams. Preferredantineoplastic agents are doxorubicin, cisplatin, and 5-fluorouracil.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically represents the effectiveness of a two stage treatmentof Brucella canis infections in mice using SPLV-entrapped streptomycinbased on B. canis recoverable from spleens of infected mice.

FIG. 2 graphically represents the effectiveness of a two stage treatmentof B. canis infections in mice using SPLV-entrapped streptomycin basedon B. canis recoverable from organs of infected mice.

FIG. 3 graphically represents the effectiveness of a two stage treatmentof Brucella abortus in guinea pigs using SPLV-entrapped streptomycin.

DETAILED DESCRIPTION OF THE INVENTION

Liposomes are completely closed lipid bilayer membranes containing anentrapped aqueous volume. Liposomes may be unilamellar vesicles(possessing a single bilayer membrane) or multilameller vesicles(onion-like structures characterized by multiple membrane bilayers, eachseparated from the next by an aqueous layer). The bilayer is composed oftwo lipid monolayers having a hydrophobic "tail" region and ahydrophilic "head" region. The structure of the membrane bilayer is suchthat the hydrophobic (nonpolar) "tails" of the lipid monolayers orienttoward the center of the bilayer while the hydrophilic "head" orienttowards the aqueous phase.

The original liposome preparation of Bangham, et al. (J. Mol. Biol.,1965, 13:238-252) involves suspending phospholipids in an organicsolvent which is then evaporated to dryness leaving a phospholipid filmon the reaction vessel. Next, an appropriate amount of aqueous phase isadded, the mixture is allowed to "swell," and the resulting liposomeswhich consist of multilamellar vesicles (MLVs) are dispersed bymechanical means. This technique provides the basis for the developmentof the small sonicated unilamellar vesicles described by Papahadjopouloset al. (Biochim. Biophys, Acta., 1968, 135:624-638), and largeunilamellar vesicles.

Unilamellar vesicles may be produced using an extrusion apparatus by amethod described in Cullis et al., PCT application No. WO 86/00238,published Jan. 16, 1986, entitled "Extrusion Technique for ProducingUnilamellar Vesicles" incorporated herein by reference. Vesicles made bythis technique, called LUVETS, are extruded under pressure through amembrane filter.

Another class of liposomes are those characterized as havingsubstantially equal intralamellar solute distribution. This class ofliposomes is denominated as stable plurilamellar vesicles (SPLV) asdefined in U.S. Pat. No. 4,522,803 to Lenk, et al., monophasic vesiclesas described in U.S. Pat. No. 4,558,578 to Fountain, et al. and frozenand thawed multilamellar vesicles (FATMLV) wherein the vesicles areexposed to at least one freeze and thaw cycle; this procedure isdescribed in Bally et al., PCT Publication No. 87/00043, Jan. 15, 1987,entitled "Multilamellar Liposomes Having Improved TrappingEfficiencies". The above noted references are incorporated herein byreference.

As used herein, liposome form will be understood to be an expansive termincluding, along with the above noted liposomes, lipid aggregates, lipidvesicles, and lipid-therapeutic agent complex.

A variety of sterols and their water soluble derivatives have been usedto form liposomes; see specifically Janoff et al., PCT Publication No.WO 85/04578, published Oct. 24, 1985, entitled "Steroidal Liposomes."Mayhew et al., PCT Publication No. WO 85/00968, published Mar. 14, 1985,described a method for reducing the toxicity of drugs by encapsulatingthem in liposomes comprising alpha-tocopherol and certain derivativesthereof. Also, a variety of tocopherols and their water solublederivatives have been used to form liposomes, see Janoff et al., PCTPublication No. WO 87/02219, published Apr. 23, 1987, entitled "AlphaTocopherol-Based Vesicles", corresponding to U.S. Pat. No. 4,861,580,issued Aug. 29, 1989.

In the present invention, the term lipid as used herein shall mean anysuitable material resulting in a bilayer such that a hydrophobic portionof the lipid material orients toward the interior of the bilayer while ahydrophilic portion orients toward the aqueous phase. Lipids furtherinclude highly hydrophobic compounds such as triglycerides, sterols suchas cholesterol which can be incorporated into the bilayer. The lipidswhich can be used in the liposome formulations of the present inventionare the phospholipids such as phosphatidylcholine (PC),phosphatidylethanolamine (PE), phosphatidylserine (PS),phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol(PI), sphingomyelin (SPM), and the like, alone or in combination. Thephospholipids can be synthetic or derived from natural sources such asegg or soy. Useful synthetic phospholipids aredymyristoylphosphatidylcholine (DMPC) anddimyristoylphosphatidylglycerol (DMPG). The liposomes can also containother steriod components such as polyethylene glycol derivatives ofcholesterol (PEG-cholesterols), coprostanol, cholestanol, or cholestane,and combinations of PC and cholesterol. They may also contain organicacid derivatives of sterols such as cholesterol hemisuccinate (CHS), andthe like. Organic acid derivatives of tocopherols may also be used asliposome-forming ingredients, such as alpha-tocopherol hemisuccinate(THS). Both CHS-and THS-containing liposomes and their tris salt formsmay generally be prepared by any method known in the art for preparingliposomes containing these sterols. In particular, see the procedures ofJanoff, et al., PCT Publication No. WO 85/04578, published Oct. 24,1985, entitled "Steroidal Liposomes," and Janoff, et al., PCTPublication No. WO 87/02219, published Apr. 23, 1987, entitled"Alpha-Tocopherol Based Vesicles," filed Sept. 24, 1986, respectively.The liposomes may also contain glycolipids.

Virtually any bioactive compound can be entrapped within a SPLV(entrapped is defined as entrapment within the aqueous compartment orwithin the membrane bilayer). Such compounds include but are not limitedto antibacterial compounds. When placed in a buffer containing isotonicsaline at neutral pH, SPLVs containing antibiotic are stable for morethan four months, as demonstrated in Table II. These data indicate thatnone of the antibiotic originally encapsulated within the SPLVs leakedout in the period of the experiment.

TREATMENT OF PATHOLOGIES

A number of pathological conditions which occur in man, animals andplants may be treated more effectively by encapsulating the appropriatecompound or compounds in SPLVs. These pathologic conditions include butare not limited to infections (intracellular and extracellular), cysts,tumors and tumor cells, allergies, etc.

Many strategies are possible for using SPLVs in the treatment of suchpathologies, a few overall schemes are outlined below which areparticularly useful in that they take advantage of the fact that SPLVswhen administered in vivo are internalized by macrophages.

In one scheme, SPLVs are used to deliver therapeutic agents to sites ofintracellular infections. Certain diseases involve an infection of cellsof the reticuloendothelical system, e.g., brucellosis. Theseintracellular infections are difficult to cure for a number of reasons:(1) because the infectious organisms reside within the cells of thereticuloendothelial system, they are sequestered from circulatingtherapeutic agents which cannot cross the cell membrane intherapeutically sufficient concentrations, and, therefore, are highlyresistant to treatment (2) often the administration of toxic levels oftherapeutic agents are required in order to combat such infections; and(3) the treatment has to be completely effective because any residualinfection after treatment can reinfect the host organism or can betransmitted to other hosts.

According to one mode of the present invention, SPLVs containing anappropriate biologically active compound are administered (preferablyintraperitoneally or intravenously) to the host organism or potentialhost organism (e.g., in animal herds, the uninfected animals as well asinfected animals may be treated). Since phagocytic cells internalizeSPLVs, the administration of an SPLV-encapsulated substance that isbiologically active against the infecting organism will result indirecting the bioactive substance to the site of infection. Thus, themethod of the present invention may be used to eliminate infectioncaused by a variety of microorganisms, bacteria, parasites, fungi,mycoplasmas, and viruses, including but not limited to: Brucella spp.,Mycobacterium spp., Salmonella spp., Listeria spp., Francisella spp.,Histoplasma spp., Corynebacterium spp., Coccidiodes spp. and lymphocyticchoriomeningitis virus.

The therapeutic agent selected will depend upon the organism causing theinfection. For instance, bacterial infections may be eliminated byencapsulating an antibiotic. The antibiotic can be contained within theaqueous fluid of the SPLV and/or inserted into the vesicle bilayer.Suitable antibiotics include but are not limited to: penicillin,ampicillin, netacillin, carbencillin, tetracycline, tetracyclinehydrochloride, oxtetracycline hydrochloride, chlortetracyclinehydrochloride, 7-chloro-6-dimethyltetracycline, doxycycline monohydrate,methacycline hydrochloride, minocycline hydrochloride, rolitetracycline,dihydrostreptomycin, streptomycin, gentamicin, kanamycin, neomycin,erythromycin, carbomycin, oleandomycin, troleandomycin, Polymysin Bcollistin, cephalothin sodium, cephaloridine, cephaloglycin dehydrate,and cephalexin monohydrate.

We have demonstrated the effectiveness of such treatments in curingbrucellosis (see Examples, infra). By the procedure of this invention,the effectiveness and duration of action are prolonged. It is surprisingthat this system is effective for treating infections which do notrespond to known treatments such as antibiotics entrapped in MLVs.Successful treatment is unexpected since any small remaining infectionswill spread and the infectious cycle will commence again. We have alsodemonstrated success in treatment lymphocytic choriomeningitis virusinfection.

Of course, the invention is not limited to treatment of intracellularinfections. The SPLVs can be directed to a variety of sites of infectionwhether intracellular or extracellular. For instance, in anotherembodiment of the present invention, macrophages are used to carry anactive agent to the site of a systemic extracellular infection.According to this scheme, SPLVs are used to deliver a therapeuticsubstance to uninfected macrophages by administering the SPLVs in vivo(preferably intraperitoneally or intravenously). The macrophages willcoalesce with the SPLVs and then become "loaded" with the therapeuticsubstance; in general, the macrophages will retain the substance forapproximately 3 to 5 days. Once the "loaded" macrophages reach the siteof infection, the pathogen will be internalized by the macrophages. As aresult, the pathogen will contact the therapeutic substance containedwithin the macrophage, and be destroyed. This embodiment of theinvention is particularly useful in the treatment of Staphylococcusaureus mastitis in man and cattle.

If the site of infection or affliction is external or accessible theSPLV-entrapped therapeutic agent can be applied topically. Aparticularly useful application involves the treatment of eyeafflictions. In the case of ocular afflictions, SPLVs containing one ormore appropriate active ingredients may be applied topically to theafflicted eye. A number of organisms cause eye infections in animals andman. Such organisms include but are not limited to: Moraxella spp.,Costridium spp., Corynebacterium spp., Diplococcus spp., Flavobacteriumspp., Hemophilus spp., KIebsiella spp., Leptospira spp., Mycobacteriumspp., Neisseria spp., Propionibacterium spp., Proteus spp., Pesudomonasspp., Serratia spp., Escherichia spp., Staphylococcus spp.,Streptococcus spp. and bacteria-like organisms including Mycoplasma spp.and Rickettsia spp. These infections are difficult to eliminate usingconventional methods because any residual infection remaining aftertreatment can reinfect through lacrimal secretions.

Aminoglycoside will be understood to mean aminoglycosides and analoguesand derivatives thereof, including streptomycin, dihydrostreptomycin,tobramycin, neomycin B, paromycin, ribostamycin, lividomycin, kanamycinA and B, viomycin, gentamicin (including C₁, C_(1a), and C₂), sisomicin,netilimicin and amikacin.

Tetracycline will be understood to mean terracyclines and analogues andderivatives thereof, including oxytetracycline, chlortetracycline,doxycycline, demeclocycline, methacycline, and minocycline.

Antineoplastic agents will be understood to include vinca alkaloids(e.g., vincristine, vinblastine), epipodophyllotoxins (e.g., etoposide,teniposide), antibiotics (e.g., dactinomycin, daunomycin, doxorubicin,bleomycin, mithramycin, mitomycin), enzymes (e.g., L-asparaginase),platinum coordinated complexes (e.g., cisplatin), substituted urea(e.g., hydroxyurea), methylhydrazine derivatives (e.g.,N-methylhydrazine,MIH), adrenocortical supressants (e.g.,aminoglutethimide), nitrogen mustards (e.g., mechlorethamine,cyclophosphamide, L-sarcolysin, uracil mustard, chlorambucil), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine,semustine, streptozocin), triazines (e.g.,dimethyltriazenoimidazolecarboxamide), folic acid analogues (e.g.,amethopterin), pyrimidine analogues (e.g., 5-flurouracil, cytosinearabinoside), purine analogues (e.g., 6-mercaptopurine, 6-thioguanine)and hormones and antagonists (e.g., prednisone, hydroxyprogesterone,medroxyprogesterone acetate, megestrol acetate, diethylstilbesterol,ethinyl estradiol, tamoxifen, testosterone propionate, fluoxymesterone).

"Therapeutic agents" shall be understood to include biologically activeagents ("bioactive agents") as well as other medically useful agentssuch as contrast materials (e.g., dyes) and diagnostic materials.

Depending upon the purpose of delivery, the SPLVs may be administered bya number of routes: in man and animals this includes but is not limitedto injection (e.g., intravenous, intraperitoneal, intramuscular,subcutaneous, intraauricular, intramammary, intraurethrally, etc.),topical application (e.g., on afflicted areas), and by absorptionthrough epithelial or mucocutaneous linings (e.g., ocular epithelia,oral mucosa, rectal and vaginal epithelial linings, the respiratorytract linings, nasopharyngeal mucosa, intestinal mucosa, etc.); inplants and protists this includes but is not limited to directapplication or organism, dispersion in the organism's habitat, additionto the surrounding environment or surrounding water, etc.

The therapeutic agent selected will depend upon the organism causing theinfection. For instance, bacterial infections may be eliminated byencapsulating an antibiotic. The antibiotic can be contained within theaqueous fluid of the SPLV and/or instead into the vesicle bilayer.Suitable antibiotics include tetracycline, tetracycline hydrochloride,oxytetracycline hydrochloride, dihydrostreptomycin, streptomycin,gentamicin, kanamycin, neomycin, erythromycin, carbomycin, oleandomycin,troleandomycin, polymixin B collistin, cephalothin sodium,cephaloridine, cephaloglycin dihydrate, and cephalexin monohydrate.

We have demonstrated the effectiveness of such treatments in curingbrucellosis (see Examples, infra). By the procedure of this invention,the effectiveness and duration of action are prolonged.

In a series of experiments (Table I) involving 28 cows, it wasdetermined that the administration of liposomal aminoglycoside was aneffective treatment for brucellosis in animals. Eight of the treatedcows were cured of brucellosis. All of the cows thus cured received anaminoglycoside (streptomycin) in liposome form. While previouslyreported work required about 12 doses of therapeutic agent, in theinstant method in particular cases less than 12 doses and, in thepreferred embodiments (including the intramammary infusion of liposomalaminoglycoside), as few as 2 intramammary doses and 2 adjunct doses.Further more in the most preferred method, the 4 doses were of the moreeasily administered type; two intramammary doses of liposomeaminoglycoside and two intramuscular doses of long-actingoxytetracycline.

The intramammary doses stated are for cows but are easily translatableto other animals relative to the size of the animal. For streptomycin adosage of about 20 to about 75 mg/kg may be used with about 35 to about50 mg/kg prefered. For various animals and therapeutic agents therelative size of the animal is considered. Adult cows, depending onbreed, are from about 800 to about 1500 pounds, adult sheep are fromabout 75 to 150 pounds and adult goats are from about 50 to 100 pounds.Intramammary doses for such animals will be generally adjusted relativeto the body weight of the animal as compared the size of a cow. Thusgoats will require a dose roughly about 1/8 to 1/30 of that of a cow andsheep roughly about 1/7 to 1/20. Similar adjustments are required forother animals such as camels, reindeer, water buffalo, and horses. Sosaying, however, a further adjustment of intramammary dose may berequired to account for the relative size of the udder. In situations inwhich exact dosages are required, a biopsy of mammary tissue or proximalmammary lymph node may be performed to ascertain the level oftherapeutic agent present. The relative doses will be understood bythose skilled in the art to be similar in relation to other therapeuticagents as compared in dosage to a dosage for cattle.

"Intramammary infusion" shall be understood to mean introducing materialinto the mammary tissue of an animal, primarily into the teat cisternand the associated afferent lymphatic system. This is done convenientlyby way of the teat canal, but other methods of introduction such asinjection into mammary tissue are similarly comtemplated. Administrationmay be to one or all teats of an animal. For clarity, reference tonumbers of treatments or administrations does not refer to the number ofteats treated but to the number of instances of treatment of the animal.

Proximal mammary lymph nodes will be understood to mean those lymphnodes within the mammary gland and those which receive lymph from themammary gland.

In a liposome-drug delivery system, a therapeutic agent such as a drugis entrapped in or associated with the liposome and then administered tothe patient to be treated. For example, see Rahman et al., U.S. Pat. No.3,993,754; Sears, U.S. Pat. No. 4,145,410; Paphadjopoulos et al., U.S.Pat. No. 4,235,871; Schneider, U.S. Pat. No. 4,114,179; Lenk et al.,U.S. Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.

The mode of administration of the preparation may determine the sitesand cells in the organism to which the compound will be delivered.Liposomes can be administered alone but will generally be administeredin admixture with a pharmaceutical carrier selected with regard to theintended route of administration and standard pharmaceutical practice.The preparations may be injected parenterally, for example,intraperitoneally, intra-arterially or intravenously. The preparationsmay also be administered via oral, subcutaneous, intramuscular and, ofcourse, intramammary routes. For parenteral administration, they can beused, for example, in the form of a sterile aqueous solution which maycontain other solutes, for example, enough salts or glucose to make thesolution isotonic. Other uses, depending upon the particular propertiesof the preparation, may be envisioned by those skilled in the art.

For administration to animals including humans in the curative treatmentof disease states, the prescribing medical professional will ultimatelydetermine the appropriate dosage for a given subject, and this can beexpected to vary according to the age, weight, and response of theanimal as well as the nature and severity of the disease. The dosage oftherapeutic agent in liposomal form will generally be about thatemployed for the free therapeutic agent. In some cases, however, it maybe necessary to administer doses outside these limits.

Aminoglycoside in liposome form will cure Brucella abortus. However,especially with large dairy animals, some methods of drug administrationsuch as intraperitoneal and intravenous are impractical, costly and (duethe unruliness of the animals) potentially dangerous to the individualadministering the treatment. Intramammary infusion is a far simplermethod by which to administer therapeutic agent. Equipment alreadyavailable in the art may be utilized to insert therapeutic agent throughthe teat canal. Further and unlike intraperitoneal administration, cleanbut not sterile conditions are usually suitable for intramammaryadministration.

As presented here, intramammary infusion of aminoglycoside in liposomeform is an effective method to treat livestock infected with Brucellaabortus.

This treatment may be effective in as few as about two to fiveapplications. In the preferred embodiment it is supplemented withco-administration of long-acting oxytetracycline, usually at about 10 to30 mg/kg animal weight and preferably about 20 mg/kg.

In one embodiment stable plurilamellar vesicles (SPLV) are the liposomesused but other types of liposomes are also contemplated. SPLV's ofphosphatidylcholine are of particular use in this invention.

The treatment via intramammary infusion of liposomes additionallypermits the treatment of the mammary tissue and proximal mammary lymphnodes of an animal for other medical conditions. This treatement isessentially perfusion (or retrograde perfusion) of the lymph system.Liposome forms may be administered to the lymph system directly as inintramammary infusion, or indirectly such as by subcutaneous orintraperitonal administration of liposome forms that will then collectin the lymph nodes. For mammary glands administration is accomplished ina fashion similar to brucellosis treatment by incorporating at least onetherapeutic agent such as an antibacterial agent, antiviral agent,antiparasitic agent, or antifungal agent (collectively "anti-infectiveagent") antineoplastic agents or other therapeutic agent into a liposomeform and administering such liposome form alone or in combination withadditional therapeutic agents to an animal. Tests of this method withthe dye Texas red indicate that liposomes introduced into the teatcistern perfuse the mammary tissue and congregate in the proximal lymphnodes in about 24 hours. Anti-infectives such as imidazoles,aminoglycosides and β-lactams, as well as antineoplastics are useful insuch method. Dosage will be similar to the dosage of free drug given ina localized manner.

Imidazole will be understood to refer to imidazoles including, withoutlimitation, miconazole, terconazole, biconazole, ketaconazole,econazole, clotrimazole and metronidazole as well as analogs andderivatives thereof characterized in having anti-infective properties.

β-lactams will be understood to refer to synthetic, semisynthetic andnatural penicillins, cephalosporins, monobactams, and thinamycins, suchas oxacillin, cephapirin, aztreonam and imipenem.

Preparation and testing of materials of the invention are describedbelow.

LIPOSOMES

The aminoglycoside liposomes were prepared as streptomycin sulfatecontaining liposomes. One process used employed egg phosphatidylcholineto make SPLVs, while another was the "emulsion-inversion process thoughother methods of liposome preparation will also be suitable.

The emulsion inversion process comprised creating a water-in-oilemulsion wherein the aqueous solution consisted of, in the case ofstreptomycin sulfate, 480 mg/ml of streptomycin sulfate in normal salineand the oil consisted of a lipid, here dried phosphatidylcholine(lecithin). In the preferred process small (about 100 ml) aliquots ofthe aqueous solution were added to about 500 gm of lecithin and blendeduntil thoroughly mixed. The process requires that in the first suchaqueous addition the total quantity of water added to the lipid phase beinsufficient to fully hydrate the lipid--thus yielding the water-in-oilemulsion. As successive aliquots of aqueous phase are added the amountof water available exceeds that amount necessary to fully hydrate thelipid and the mixture inverts into an oil-in-water emulsion. At thispoint the hydrated lipids have become liposomes. Since encapsulation oftherapeutic agent--here streptomycin--occurs prior to full hydrationtherapeutic agent was added only for the pre-full hydration additions ofaqueous solution. In this example this was the first three aqueousadditions. Of course the nature of the particular lipid as well as itsstate of dehydration will vary the amounts of aqueous phase for thepre-and post-hydration stages. The exact proportions will be clear tothose skilled in the art as delineated by the stage of hydration atwhich liposomes appear.

The resulting liposomes were suspended in 0.9% saline. In some cases,the liposomes were reduced in size by passing them through a 0.5 micronorifice under pressure. The total and entrapped streptomycin content wasdetermined by high performance liquid chromatographic procedure (Wall,T. J., "Determination of Streptomycin Sulfate by High Performance LiquidChromatography", J Chromatography, 219:89-100, (1981)) or an agar-welldiffusion assay using Antibiotic Assay Medium #5 (BBL; Baltimore, Md.)and Bacillus subtilis (ATCC #6633) as an indicator organism. Theparticular assay method is not critical and other methods will be knownto those skilled in the art.

The total phospholipid phosphate was determined using a modifiedBartlett's assay (Rouser, G., et al., Lipids 5:494, 1970).

Lipid composition was qualtitatively assessed by thin layerchromatography. Vesicle size was estimated using a Nicomp (Hiac/Royco;Goleta Calif.) Model 270 sub-micron particle sizes for vesicles lessthan 1 micron in diameter and either a Brinkman (Brinkman Instruments,Westbury, N.Y.) Particle Size analyzer or a Malvern (MalvernInstruments, Malvern England) Particle Sizer for vesicles in the rangeof 1-30 microns diameter.

The liposomes produced by either SPLV or emulsion-inversion entrappedabout 26 to 80% of the aminoglycoside present. The exogenousaminoglycoside can be washed free of the liposomes but was not in theexamples reported here.

BACTERIOLOGICAL STUDIES

Pre-treatment cultures of udder secretions were made to confirm sheddingof Brucella abortus by subject animals. Samples for culture werecollected every 3 to 4 days during therapy and for 3 to 6 weeks afterthe last treatment. Cows were slaughtered and the selected tissues (thesupramammary, iliac and head lymph nodes and the udders) were collected,processed in a tissue homogenizer and plated.

All samples were plated on trypose agar containing crystal violet andincubated at 37° C. with 5% CO₂ for 5 to 7 days.

Animals from which Brucella could no longer be recovered from uddersecretions nor from any tissues examined at necropsy were consideredcured.

TREATMENT PROTOCOLS

The treatment of the test animals employed, in various combinations, 4techniques:

(1) intraperitoneal injection of aminoglycoside liposomes;

(2) intravenous injection of aminoglycoside liposomes;

(3) intramammary infusion of aminoglycoside liposomes; and

(4) intramuscular injection of a tetracycline.

Intraperitoneal (IP) injections of streptomycin sulfate in liposome formutilizing liposomes with either 59 mg/kg to 118 mg/kg animal weight oftherapeutic agent were administered per dose in a volume ofapproximately 360 ml at 3-4 day intervals.

Intravenous injections of aminoglycoside in liposome form wereadministered at 20 mg/kg animal weight in a volume of 180 to 400 ml perdose at 3 day intervals. Intervals of from about 1 to 5 days are alsoacceptable.

Intramammary infusion utilized 50 ml of aminoglycoside in liposome formper udder quarter at 1-3 day intervals. Intervals up to about 5 days arealso acceptable. Infusion was accomplished by inserting a teat cannulain the teat canal and injecting the liposome preparation through a buntneedle into the teat cistern.

Intramuscular injection of tetracycline employed oxytetracycline in apyrrolidine vehicle (LA-200™) which will be referred to as long-actingoxytetracycline. Long-acting oxytetracycline was used in a solution of200 mg/ml administered intramuscularly in the cervical region at adosage of 20 mg/kg animal weight injecting 10-15 ml per cite at 3 to 4day intervals. Intervals of from about 1 to 6 days are also acceptable.

EXAMPLE 1 TABLE I(A), INTRAMAMMARY AND INTRAPERITONEAL

Two cows naturally infected with Brucella abortus were used. The firstcow weighed 500 kg and received intraperitoneal streptomycin sulfate inliposome form (emulsion-inversion liposomes) in 2 treatments spaced 3days apart of 325 ml of liposomes, and a dosage of 59 mg/kg with thetotal streptomycin administered intraperitoneally being 60 gm. This cowalso received 4 intramammary infusions of streptomycin sulfate inliposome form.

These intramammary infusions were into udder quarters previouslydetermined to be shedding Brucella. The 4 intramammary infusions wereeach of 3.75 gm of streptomycin sulfate in 50 ml of liposome andtotalled 15 gms. These 4 infusions where at 2 to 3 day intervals.

The second cow weighed 420 kg and also received 59 mg/kg animal weightintraperitoneal streptomycin sulfate in liposome form in 2 treatmentseach in a volume of 270 ml at 3 day intervals for a total of 50 gmsstreptomycin sulfate. This cow was determined to be shedding from twoudder quarters and received an intramammary infusion into each quarterof 7.5 gm of liposome form streptomycin sulfate each quarter receiving50 ml volumes of liposomes. This cow received therapeutic agent only inthe 2 positive quarters for 3 of the 4 doses administered, and oneadministration to all 4 quarters. Total intramammary administration was76 gms of streptomycin sulfate in liposome form.

Both cows were cured in 6 total treatments by the combined use ofintraperitoneal aminoglycoside liposomes and intramammary infusion ofaminoglycoside liposomes.

EXAMPLE 2 TABLE I(B), INTRAPERITONEAL, INTRAMUSCULAR

Two cows determined to be shedding Brucella abortus were utilized. Thefirst weighed 600 kg and received intraperitoneal streptomycin sulfatein liposome form (emulsion-inversion liposomes) in 5 treatments each of385 ml of liposomes at 3 to 4 day intervals and a dosage of 59 mg/kgwith total streptomycin administered intraperitoneally being 177 gms.This cow also received 5 treatments of long-acting oxytetracyclineintramuscularly in doses of 20 mg/kg at 3 to 4 day intervals.

In similar fashion a second cow of 490 kg received 5 doses ofintraperitoneal streptomycin sulfate in liposome form(emulsion-inversion liposomes) in volumes of 315 ml per dose at 3 to 4day intervals. Dosage was at 50 mg/kg for a total of 145 gm totalstreptomycin. Five doses of long-acting oxytetracycline were alsoadministered intramuscularly at 20 mg/kg each dose in 3 to 4 dayintervals.

In 10 doses (none of which were the more easily administeredintramammary infusion) both animals were cured.

EXAMPLE 3 TABLE I(C), INTRAVENOUS, INTRAMUSCULAR

Two cows determined to be shedding Brucella abortus were utilized. Thefirst cow weighed 580 kg and received 2 intravenous doses ofstreptomycin sulfate of 400 ml in liposome form (SPLV liposomes) in 3day intervals. Dosage was at 75 mg/kg for at total of 86 gms. Thisanimal also received intramuscular long-acting oxytetracycline at 20mg/kg in two doses at 3-4 day intervals. This animal was cured.

The second cow weighed 580 kg received exactly the same treatment andwas not cured.

EXAMPLE 4 TABLE I(D), INTRAMAMMARY, INTRAMUSCULAR

This example utilized 5 cows two of which were cured. Due to the ease oftreatment utilizing the relatively simple intramammary infusion ofliposome form (SPLV liposomes) aminoglycoside supplemented withlong-acting oxytetracycline this is the preferred method.

All five cows received two intramuscular doses of long actingoxytetracycline at 20 mg/kg in 3 to 4 day intervals.

Two cows received 2 treatments at 3 day intervals of 5.4 gm ofstreptomycin sulfate by intramammary infusion into each quarter of theudder in 50 ml of liposomes for a total of 43 gms of streptomycin. Oneof these cows was cured in the 4 treatments. Three cows received thesame treatment except that 3.5 gm of streptomycin sulfate liposomes wereused in each udder quarter for a total of 28 gms. One of the three cowswas cured in the 2 intramammary treatments accompanied by the 2intramuscular treatments.

EXAMPLE 5 TABLE I(E), INTRAPERITONEAL

Six cows weighing from 396 to 508 kg shedding Brucella abortus wereused. Each cow received 5 intraperitoneal doses of streptomycin sulfatein liposome form (4 receiving emulsion-inversion liposomes and 2receiving SPLV liposomes) in 1 to 3 day intervals and no othertreatment.

Streptomycin sulfate was administered to 2 cows at 59 gm/kg, 2 cows at62 mg/kg and 2 cows at 118 mg/kg. One cow, weighing 403 kg receiving 5doses 62 mg/kg in 330 ml in liposome form (SPLV) for a total of 123 gmsof streptomycin, was cured.

EXAMPLE 6 TABLE I(F), INTRAVENOUS

Two cows shedding Brucella abortus were used. Each cow weighed 580 kgand received streptomycin sulfate in liposome form in 2 doses of 1 to 3day intervals. Dosage was at 75 mg/kg and 400 ml in liposome form (SPLVliposomes) was used each dose. Total streptomycin sulfate administeredwas 86 gms with no cure.

EXAMPLE 7 TABLE I(G), INTRAVENOUS, INTRAMAMMARY

Three cows shedding Brucella abortus were used. Each cow weighed 625 kgand each received two doses of 20 mg/kg and each received two doses of20 mg/kg streptomycin sulfate in liposome form (SPLV liposomes) at 1 to3 day intervals for a total of 25 gms of streptomycin sulfate. Each dosewas 180 ml of liposome. Each cow also received 2 treatments ofstreptomycin sulfate in liposome form at 3.5 gms per udder quarter, 50ml of liposome, per treatment for a total of 28 gms. No cows were cured.

EXAMPLE 8 TABLE I(H), INTRAMAMMARY

Six cows shedding Brucella abortus were used. Three cows received asingle treatment of streptomycin sulfate in liposome form (SPLVliposomes). Treatment was to two of the udder quarters on each cow in 50ml volumes of liposomes. One cow received 0.96 gms per udder quartertreated, one received 1.93 gms per udder quarter treated, and onereceived 3.85 gms per udder quarter treated.

Of the other three cows, one received two doses by intramammary infusionof streptomycin sulfate in 50 ml in liposome form (SPLV liposomes), eachtreatment being 0.96 gm streptomycin sulfate per udder quarter to all 4quarters. Doses were at an interval of 3 days. One received 4 doses byintramammary infusion of streptomycin sulfate in 50 ml in liposome form,each treatment being 1.93 gm streptomycin sulfate per udder quarter toall 4 quarters at an interval of 3 days. On receiving 2 doses byintramammary infusion of streptomycin sulfate in 50 ml in liposome form,each treatment being 3.85 gm streptomycin sulfate per udder quarter toall 4 quarters at an interval of 3 days. No animals were cured.

EXAMPLE 9 SPLVS CONTAINING ANTIBIOTICS

A 5 ml diethyl ether solution of 100 mg lecithin ws prepared. Themixture was placed in a round-bottom flask. Then a solution (0.3 ml)containing 100 mg of streptomycin sulfate at pH 7.4 in 5 mM HEPES(4-[2-Hydroxyethyl]piperazino 2-ethane sulfonic acid)/0.0725MNaCl/0.0725M KCl was pipetted into the glass vessel containing thediethyl ether solution of lipid. The mixture was placed in a bathsonicator (Laboratory Supplies Co., Inc.) type 10536 for severalminutes, (80 kH_(z) frequency:output 80 watts) while being dried to aviscous paste by passing thereover a gentle stream of nitrogen.

To the viscous paste remaining was added 10 ml of 5 mM HEPES. Theresulting SPLV preparation, containing streptomycin, was suspended inthe buffer solution, shaken for several minutes on a vortex mixer, andfreed of non-encapsulated streptomycin by centrifuging at 12,000×g for10 minutes at 20° C. The resulting cake was suspended in 0.5 ml of 5 mMHEPES.

The procedure described above was followed except that streptomycin wassubstituted by each one of the following: dihydrostreptomycin,gentamycin sulfate, ampicillin, tetracycline hydrochloride, andkanamycin.

EXAMPLE 10 SPLV Mediated Delivery In Vitro

In the following example, SPLV mediated delivery of antibiotics tomacrophages in culture was demonstrated.

Peritoneal macrophages were obtained by peritoneal lavage from C₅₇ BLKadult male mice and suspended in minimal essential medium (M.E.M.) pH7.2 containing 10% heat-inactivated fetal calf serum. Cells weresuspended at a concentration of 1×10⁶ cells per ml in 96-well tissueculture dishes. To cultures containing adherent peritoneal macrophages,were added B. canis at concentrations of 1×10⁶ CFU (colony formingunits) per ml. After 12 hours, bacteria not engulfed by peritonealmacrophages were removed by repeated washings with M.E.M. After washingof peritoneal macrophage cultures, they were divided into 5 groups, eachcontaining 12 replicate cultures per group. Group 1, designatedControls, received no treatment. Group 2 received aqueous streptomycinsulfate at a concentration of 1 mg/ml. Group 3 received buffer-filledSPLVs. Group 4 received aqueous streptomycin sulfate (1 mg/ml) pluspreformed buffer-filled SPLVs. Group 5 received SPLVs containingstreptomycin sulfate (1 mg/ml). After 24 hours, supernatants wereremoved by repeated washings and peritoneal macrophages were disruptedby repeated freezing and thawing. Serial dilutions of disruptedmacrophages were plated onto brucella agar and, after 4 days, survivingB. canis were determined by limiting dilution techniques. Results shownin Table VIII indicate that SPLV-entrapped streptomycin was totallyeffective in killing and eliminating the intracellular B. canisinfection in vitro.

The experiment was repeated with B. abortus exactly as described aboveexcept that peritoneal macrophages were obtained by peritoneal lavagefrom adult female albino guinea pigs. Results are also shown in TableVIII.

EXAMPLE 11 Treatment Intracellular Infections

The following examples demonstrate how SPLVs can be used in treatingintracellular infections. The data presented demonstrates: (1) theeffectiveness of using antibiotics encapsulated in SPLVs in thetreatment of disease and (2) the greater efficiency which is obtained byadministering multiple doses of the SPLV preparation.

Brucellosis causes worldwide economic and public health problems.Brucellosis is caused by Brucella spp. It is adapted to many mammalianspecies, including man, domestic animals and a variety of wild animals.Six Brucella spp. cause brucellosis in animals; they are B. abortus, B.canis, B. melitensis, B. neotomae, B. ovis and B. suis. Both domesticand wild animals serve as reservoirs for potential spread of brucellosisto other animals and man.

Such infections cannot be cleared with antibiotics because theinfectious organisms reside within the cells of the reticuloendothelialsystem and are highly resistant to bactericidal activities ofantibiotics. The quantity of antibiotics required and the length oftreatment results in either toxic effects on the animal or anunacceptable high concentration of the antibiotic in the tissues of theanimal. The further difficulty in treating this disease is that thetreatment has to be completely effective since any remaining infectionwill simply spread and the cycle commences once again. The economicimpact of such diseases is demonstrated by the millions of dollars ofvaluable cattle which are lost each year due to spontaneous abortion.The only potential way to combat such infectious outbreaks is toquarantine and then slaughter the animals.

The examples which follow comprise incorporating an antibiotic intoSPLVs, and then administering the encapsulated active substance to theanimals by inoculating the infected animals intraperitoneally.

Effect of a Single Treatment of B. Canis Infection Using SPLV-EntrappedAntibiotic

Eighty adult male Swiss mice were infected intraperitoneally (O.P.) withB. canis ATCC 23365 (1×10⁷ CFU) and divided into 8 groups of 10 miceeach. Seven days post-inoculation with B. canis groups were treated asfollows: Group 1, designated Controls, received no treatment; Group 2received buffer-filled SPLVs (0.2 ml I.P.); Group 3 received aqueousstreptomycin sulfate (1 mg/kg body weight in a total administration of0.2 ml I.P.); Group 4 received aqueous streptomycin sulfate (5 mg/kgbody weight) in a total administration of 0.2 ml I.P.; Group 5 receivedaqueous streptomycin sulfate (10 mg/kg body weight) in a totaladministration of 0.2 ml I.P.; Group 6 received SPLVs containingstreptomycin sulfate (1 mg/kg body weight) in a total administration of0.2 ml I.P.; Group 7 received SPLVs containing streptomycin sulfate (5mg/kg body weight) in a total administration of 0.2 ml I.P.; and Group 8received SPLVs containing streptomycin sulfate (10 mg/kg body weight) ina total administration of 0.2 ml I.P. On day 14 post-inoculation with B.canis, all animals were sacrificed and spleens were removed aseptically.Spleens were homogenized and serially diluted onto brucella agar todetermine the number of surviving B. canis in spleens after treatment.Results after 4 days incubation are shown in Table IV.

EXAMPLE 12 Effect of Multiple Treatment of B. Canis Infection UsingSPLV-Entrapped Antibiotic

Eighty adult male Swiss mice were infected with B. canis ATCC 23365(1×10⁷ CFU, I.P.) and divided into 8 groups of 10 mice each. Seven and10 days post-inoculation with B. canis groups were treated as follows:Group 1, designated Controls, received no treatment; Group 2 receivedbuffer-filled SPLVs (0.2 ml I.P.); Group 3 received aqueous streptomycinsulfate (1 mg/kg body weight in a total administration of 0.2 ml I.P.);Group 4 received aqueous streptomycin sulfate (5 mg/kg body weight) in atotal administration of 0.2 ml I.P.; Group 5 received aqueousstreptomycin sulfate (10 mg/kg body weight) in a total administration of0.2 ml I.P.; Group 6 received SPLVs containing streptomycin sulfate (1mg/kg body weight) in a total administration of 0.2 ml I.P.; Group 7received SPLVs containing streptomycin sulfate (5 mg/kg body weight) ina total administration of 0.2 ml I.P.; and Group 8 received SPLVscontaining streptomycin sulfate (10 mg/kg body weight) in a totaladministration of 0.2 ml I.P. On day 14 post-inoculation with B. canis,all animals were sacrificed and spleens were removed aseptically.Spleens were homogenized and serially diluted onto brucella agar todetermine the number of surviving B. canis in spleens after treatment.Results after 4 days incubation are shown in FIG. 1.

The results of various two-stage treatment regimens on B. canisinfections in vivo presented in FIG. 3, demonstrate that in groupsreceiving aqueous streptomycin 7 and 10 days post-inoculation, verylittle reduction in surviving B. canis in spleens was observed. Only ingroups receiving SPLV-entrapped streptomycin at a concentration of 10mg/kg body weight administered on day 7 and 10 post-inoculation were allviable bacterial eliminated from spleens of infected animals.

In addition to the experiment described above, various tissues from B.canis infected mice after two treatments with SPLV-entrappedstreptomycin were sampled as follows:

Thirty adult male Swiss mice were inoculated with B. canis ATCC 23365(1×10⁷ CFU, I.P.). Seven days post-inoculation animals were divided into3 groups of 10 mice each. Group 1, designated controls, received notreatment; Group 2 received (on days 7 and 10 post-inoculation) aqueousstreptomycin sulfate (10 mg/kg body weight) in each administration of0.2 ml), I.P.; Group 3 received (on days 7 and 10 post-inoculation)SPLVs containing streptomycin sulfate (10 mg/kg body weight) in eachadministration of 0.2 ml, I.P. On days 14 to 75 post-inoculation with B.canis all animals were sacrificed and the following organs removedaseptically, homogenized and serially diluted onto brucella agar forisolation of B. canis: heart, lungs, spleen, liver, kidneys, testes.After 4 days incubation, results of surviving B. canis per organ areshown in FIG. 2.

Results of samplings of various tissues in B. canis infected mice aftertwo treatment regimens with streptomycin presented in FIG. 2demonstrated that in animals treated with SPLV-entrapped streptomycin,all tissues sampled from 14 to 75 days post-inoculation with B. caniswere totally free to any viable B. canis organisms. In animals untreatedor treated with aqueous streptomycin in concentrations andadministration schedules identical to those receiving SPLV-entrappedstreptomycin, viable B. canis organisms could be isolated in all tissuessampled from 14 to 75 days post-inoculation with B. canis.

EXAMPLE 13 Effect of Various SPLV-Entrapped Antibiotics on Treatment ofInfection

Fifty adult male Swiss mice were inoculated with B. canis ATCC 23365(1×10⁷ CFU, I.P.). Seven days post-inoculation animals were divided into10 groups of 5 mice each. Group 1, designated controls, received notreatment; Group 2 received buffer-filled SPLVs (0.2 ml, i.P.) on days 7and 10 post-inoculation; Groups 3, 4, 5 and 6 received aqueousinjections (0.2 ml I.P.) of dihydrostreptomycin, gentamicin, kanamycinor streptomycin 10 mg/kg body weight, I.P. on days 7 and 10post-inoculation (N.B. Each of these antibiotics have been shown to killB. canis in vitro).

Groups 7, 8, 9 and 10 received SPLVs containing dihydrostreptomycin,gentamicin, kanamycin, or streptomycin at 10 mg/kg body weight on days 7and 10 post-inoculation. On day 14 post-inoculation with B. canis, allanimals were sacrificed and spleens were removed aseptically,homogenized and serially diluted onto brucella agar for at isolation ofB. canis. Results of surviving B. canis per organ after 4 daysincubation are as shown in Table V.

The results from tests of various antibiotics on B. canis infected micepresented in Table V demonstrate that antibiotics which are effective inkilling B. canis in vitro (i.e., in suspension culture) are also onlyeffective in killing B. canis infections in vivo when they areencapsulated within SPLVs. Animals receiving either aqueous antibiotics,buffer-filled SPLVs or no treatment were in no case cleared of survivingB. canis in isolated spleen tissues.

EXAMPLE 14 Treatment of Dogs Infected with B. canis

Adult female beagles were inoculated with B. canis ATCC 23365 (1×10⁷CFU) orally and vaginally. Seven days post-inoculation dogs were dividedinto 3 groups. Group 1, designated control, received no treatment; Group2 received (on days 7 and 10 post-inoculation) aqueous streptomycinsulfate at 10 mg/kg body weight (each administration was 5.0 ml, I.P.).Group 3 received (on days 7 and 10 post-inoculation) SPLVs containingstreptomycin sulfate at 10 mg/kg body weight (each administration was3.0 ml, I.P.). Vaginal swabbings of dogs and heparinized blood sampleswere collected at regular intervals before, during, and at thetermination of the study. These were cultured on brucella agar in orderto isolate B. canis. Results are shown in Table VI. Serum samples werecollected before, during, and at the termination of the study fordeterminations of serum antibody against B. canis. These results arealso shown in Table VI. Twenty-one days post-inoculation with B. canis,all animals were euthanized. The following tissues were removedaseptically, homogenized and serially diluted onto brucella agar forisolation of B. canis: heparinized blood, vaginal exudate, lungs,spleen, synovial fluid, uterus, ovary, popliteal lymph nodes, salivaryglands, tonsils, mediastinal lymph nodes, mesenteric lymph nodes, bonemarrow, superficial cervical lymph nodes, and auxiliary lymph nodes.Results of surviving B. canis per tissue after 4 days incubation areshown in Table VII.

Results of culture and serological tests of dogs infected with B. canisbefore, during, and after two-stage antibiotic administration arepresented in Table VI. All animals were serologically negative forprevious exposure to B. canis as measured by negative serum titers, andwere culture negative from blood cultures and cultures of vaginalswabbings. All animals were noted to be culture positive for both bloodand vaginal cultures prior to treatments on days 7 and 10. Dogs treatedwith aqueous streptomycin or dogs receiving no treatment remainedculture positive for bood and vaginal cultures during post-treatmentperiods prior to termination on day 21. Group 3, which receivedliposomes containing streptomycin, became culture negative one day afterthe first treatment and remained negative throughout post-treatmentperiod. Dogs which received no treatment or aqueous streptomycindeveloped detectable serum titers against B. canis antigens by day 21post-inoculation, while those treated with SPLVs containing antibioticson days 7 and 10 post-inoculation did not develop any detectableantibody to B. canis antigen.

Results from isolation of B. canis from infected dogs treated withtwo-stage antibiotic administration which are presented in Table VIIdemonstrate that in dogs, only treatment with SPLVs containingstreptomycin was effective in eliminating any viable B. canis in alltissues from all organ samples.

EXAMPLE 15 Treatment of B. abortus in Guinea Pigs

Fifteen adult female guinea pigs were inoculated with B. abortus ATCC23452 (1×10⁷ CFU, I.P.). Seven days post-inoculation animals weredivided into 3 groups of 5 animals each. Group 1, designated Controls,received no treatment. Group 2 received aqueous streptomycin sulfate,I.P. injections (0.2 ml) at 10 mg/kg body weight on day 7 and 10post-inoculation with B. abortus. Group 3 received SPLVs containingstreptomycin sulfate I.P. injections (0.2 ml) at 10 mg/kg body weight ondays 7 and 10 post-inoculation with B. abortus. On day 14post-inoculation with B. abortus, all animals were sacrificed andspleens were removed, aseptically homogenized and serially diluted ontobrucella agar for silation of B. abortus. Results of surviving B.abortus per spleen after 4 days incubation, are shown in FIG. 3. OnlySPLVs containing streptomycin were effective in eliminating B. abortusresiding within guinea pig spleen. In animals receiving aqueousstreptomycin or no treatment, viable B. abortus bacteria wereidentified.

EXAMPLE 16 Treatment of B. abortus Infection in Cows

Nine heavily infected animals were utilized in this experiment. B.abortus bacterial isolations from milk and vaginal swabbings became andremained negative for six weeks following treatment with SPLVscontaining streptomycin. When infection reoccurred in these animals,bacterial isolations were found only in quadrants of the udder whichwere positive prior to treatment.

Nine cross-bred (hereford-jersey-Brangus), 22-month old, non-gravid,confirmed B. abortus culture-positive cows were used. At least 4 monthsprior to the initiation of the study, the animals were experimentallychallenged per conjunctivum with 1×10⁷ CFU of B. abortus Strain 2308during mid-gestation, which resulted in abortion and/or B. abortusculture positive lacteal or uterine secretions and/or fetal tissues.

Cows were maintained in individual isolation stalls and separated intothree groups. Treatment comprised a two-dose regimen, spaced 3 daysapart, as follows: (1) 3 cows were injected intraperitoneally withphysiological saline. (2) 3 cows were injected intraperitoneally withaqueous antibiotic (streptomycin at 10 mg/kg body weight) plus preformedbuffer-filled SPLVs. (3) 3 dows were injected intraperitonealy withSPLV-entrapped streptomycin (10 mg/kg body weight). The total volume perinjection was 100 ml per animal.

During the first 2 months duplicate bacteriologic cultures of lactealand uterine secretions were performed weekly providing secretions wereobtainable. Then, all 5 cows were euthanized with an overdose of sodiumpentabarbitol, and the following organs were collected in duplicate forbacteriologic cultures: (1) lymph nodes: left and right atlantal, leftand right suprapharyngeal, left and right mandibular, left and rightparotid, left and right prescapular, left and right prefemoral, left andright axillary, left and right popliteal, left and right internal iliac,left and right supramammary, left and right renal, bronchial,mediastinal, mesenteric, and hepatic; (2) glands: all four quarters ofmammary gland, left and right adrenal glands and thymus (if present);(3) organs and other tissues: spleen, liver, left and right horn ofuterus, cervix, vagina, kidney and tonsil.

After necropsy, all tissues were frozen and maintained at -70° C. whilein transport. Tissues were thawed, alcohol flamed, and asepticallytrimmed prior to weighing. Once weights were recorded (0.2 to 1.0grams), the tissue was homogenized in 1 ml of sterile saline andserially diluted with sterile saline to 1:10-¹⁰ of initial homogenatesuspension. Aliquots (20 ul) of each dilution from serial suspensionswere plated onto brucella agar and placed in 30° C. incubation.Duplicate determinations were performed for each tissue.

Plates were read daily and scored for bacterial growth. All coloniesappearing prior to 3 days were isolated, passaged, and gram stained todetermine identity. On days 5, 6 and 7 during incubation colonies withmorphology, growth, and gram staining characteristics consistent with B.abortus were counted; the CFU per gram tissue was then determined.Representative colonies were repassaged for bacterial confirmation of B.abortus.

Bacteriologic isolations were done on all tissue samples andquantitation of bacteria per gram of tissue were calculated. The resultsfrom four animals--one placebo control and three animals treated withSPLV-entrapped streptomycin--are presented in Table VIII.

                  TABLE II                                                        ______________________________________                                        STABILITY OF EGG PHOSPHATIDYLCHOLINE                                          SPLVs AFTER STORAGE IN SEALED                                                 CONTAINERS AT 4° C. FOR 41/2 MONTHS.sup.a                                         Initial    Leakage    Bioavailability                              Entrapped  Entrapment Into       of Entrapped                                 Drug       %          Supernatant.sup.b                                                                        Drug (%)                                     ______________________________________                                        Streptomycin                                                                             34.1       0          97                                           Sulfate                                                                       Spectinomycin                                                                            37.2       0          84                                           Chloramphenicol                                                                          35.2       0          89                                           Oxytetracycline                                                                          18.8       0          91                                           Erythromycin                                                                             0.4        0          97                                           Sulfamerazine                                                                            6.3        0          93                                           ______________________________________                                         .sup.a SPLVs were prepared using 127 μM egg phosphatidylcholine (EPC)      and 25 μM drug. At the end of 41/2 months storage at 4° C. the      SPLVs were separated from storage buffer by centrifugation. Serial            dilutions of the SPLV contents and the supernatant were applied to            bacterial lawns in order to determine bioactivity as compared to standard     dilutions of antibiotic.                                                      .sup.b 0 indicates below detectable levels                               

                  TABLE III                                                       ______________________________________                                        COLONY-FORMING UNITS OF INTRACELLULAR                                         BRUCELLA ISOLATED AFTER TREATMENT                                             OF INFECTED MACROPHAGES WITH                                                  SPLVS CONTAINING STREPTOMYCIN                                                            B. canis.sup.a                                                                            B. abortus.sup.b                                       ______________________________________                                        Controls      2.6 ± 1.13 × 10.sup.3                                                            3.1 ± 0.81 × 10.sup.4                      Buffer-filled                                                                              2.82 ± 0.10 × 10.sup.3                                                            2.9 ± 0.17 × 10.sup.4                      SPLVs                                                                         Free         3.11 ± 0.40 × 10.sup.3                                                            3.3 ± 0.25 × 10.sup.4                      Streptomycin.sup.c                                                            Streptomycin 2.76 ± 0.20 × 10.sup.3                                                            2.8 ± 0.42 × 10.sup.4                      Plus Buffer-                                                                  filled SPLVs.sup.c                                                            SPLV-Entrapped                                                                             0            0                                                   Streptomycin.sup.c                                                            ______________________________________                                         .sup.a Colony forming units (CFU) of B. canis (mean ± SD of 12             replicates) isolated from equal numbers of previously infected mouse          (C.sub.57 Blk) peritoneal macrophages.                                        .sup.b CFU of B. abortus (mean ± SD of 12 replicates) isolated from        equal numbers of previously infected guinea pig peritoneal macrophages.       .sup.c Concentration of streptomycin 1 mg/ml.                            

                                      TABLE IV                                    __________________________________________________________________________    EFFECT OF A SINGLE TREATMENT.sup.a OF B. CANIS                                INFECTED MICE WITH VARIOUS CONCENTRATIONS                                     OF FREE OR SPLV-ENTRAPPED STREPTOMYCIN                                        __________________________________________________________________________                Colony-Forming Units B. Canis Per Spleen.sup.b                                No Treatment Buffer-Filled SPLVs                                  __________________________________________________________________________    Control     3.46 × 10.sup.6 ± 2.7 × 10.sup.6                                            4.1 × 10.sup.6 ± 0.66                       __________________________________________________________________________                             × 10.sup.6                                     Streptomycin                                                                  Concentration                                                                             Free         SPLV-Entrapped                                       (mg/kg body weight)                                                                       Streptomycin Streptomycin                                         __________________________________________________________________________    1            1.5 ± 0.45 × 10.sup.6                                                            1.01 ± 0.25 × 10.sup.3                      5           2.12 ± 1.71 × 10.sup.5                                                             1.52 ± 0.131 × 10.sup.4                    10          9.66 ± 3.68 × 10.sup.4                                                            1.32 ± 1.00 × 10.sup.4                      __________________________________________________________________________     .sup.a I.P injection in total of 0.2 ml (sterile saline).                     .sup.b Surviving B. canis was determined as the number of CFU isolated pe     spleen and is expressed as mean ± S.D. of 10 animals per experiment        (triplicate experiments).                                                

                  TABLE V                                                         ______________________________________                                        COMPARISON OF VARIOUS ANIBIOTICS ON KILLING                                   OF B. CANIS IN VIVO AFTER TWO TREATMENTS.sup.a                                            Colony-Forming Units                                                          B. Canis Per Spleen.sup.b                                                     Aqueous    SPLV-Entrapped                                                     Solutions  Antibiotic                                             ______________________________________                                        Untreated     3.93 ± 1.51 × 10.sup.6                                                            4.66 ± 0.87 × 10.sup.6                    Antibiotic.sup.c                                                              Dihydrostreptomycin                                                                         1.13 ± 0.30 × 10.sup.5                                                            0                                                  Gentamycin    7.06 ± 2.53 × 10.sup.5                                                            0                                                  Kanamycin     2.72 ± 0.91 × 10.sup.5                                                            0                                                  Streptomycin  1.01 ± 0.17 × 10.sup.5                                                            0                                                  ______________________________________                                         .sup.a Intraperitoneal treatments, 10 mg/kg body weight, were spaced at 3     day intervals. Controls received no treatment.                                .sup.b Surviving B. canis per organ was determined as the number of CFU       isolated per spleen and expressed as the mean ± S.D. of 5 animals per      groups (duplicate determinations per animal).                                 .sup.c Antibiotics effective in killing B. canis in suspension culture.  

                                      TABLE VI                                    __________________________________________________________________________    RESULTS OF CULTURES AND SERLOGICAL TESTING                                    IN B. CANIS INFECTED DOGS SUBJECTED                                           TO A TWO TREATMENT ANTIBIOTIC REGIMEN.sup.a                                   Days After                      SPLV-                                         Infection                       Entrapped                                     with    Control     Streptomycin.sup.b                                                                        Streptomycin.sup.c                            B. Canis                                                                              R  M  B  V  R  M  B  V  R  M  B  V                                    __________________________________________________________________________    Pre-treatment                                                                 0       0  0  0  0  0  0  0  0  0  0  0  0                                    2       ND ND +  +  ND ND +  0  ND ND +  +                                    4       ND ND +  +  ND ND +  +  ND ND +  +                                    Post-treatment                                                                8       0  0  0  +  0  0  +  0  0  0  0  0                                    10      0  0  0  +  0  0  0  +  0  0  0  0                                    21        1.5                                                                            2  +  +  1  2  +  +  0  0  0  0                                    __________________________________________________________________________     .sup.a R (rapid slide agglutination test) indicates the reciprocal of         serum titer to B. canis antigen (× 10.sup.2); 0 = no detectable         titer.                                                                        M (2mercaptoethanol tube agglutination test) indicates the reciprocal of      serum titer to B. canis antigen (× 10.sup.2); 0 = no detectable         titer.                                                                        In B (blood culture) and V (vaginal culture) on brucella agar: + =            detection of greater than or equal to 1 CFU; 0 = no colonies detected.        Controls received no treatment.                                               .sup.b Streptomycin sulfate (aqueous) 10 mg/kg body weight, I.P.              .sup.c SPLVs containing streptomycn sulfate 10 mg/kg body weight, I.P.   

                  TABLE VII                                                       ______________________________________                                        RESULTS OF CULTURES FROM TISSUE SAMPLES                                       IN B. CANIS INFECTED DOGS SUBJECTED                                           TO A TWO TREATMENT ANTIBIOTIC REGIMEN.sup.a                                                SPLVs                                                                         Containing                                                       Tissue.sup.b Streptomycin.sup.c                                                                       Streptomycin.sup.d                                                                        Control.sup.e                             ______________________________________                                        Whole blood  0          +           +                                         Vaginal swab 0          +           +                                         Lungs        0          +           +                                         Spleen       0          +           +                                         Synovial fluid                                                                             N.D.       0           0                                         Uterus       0          +           +                                         Ovary        0          +           +                                         Popliteal lymph node                                                                       N.D.       +           +                                         Salivary gland                                                                             0          0           0                                         Tonsil       0          +           +                                         Mediastinal lymph                                                                          0          N.D.        +                                         node                                                                          Mesenteric lymph                                                                           N.D.       0           0                                         node                                                                          Bone marrow  0          +           +                                         Superficial  N.D.       N.D.        +                                         cervical lymph node                                                           Axillary lymph node                                                                        0          +           +                                         ______________________________________                                         .sup.a Animals treated on day 7 and 10 postinfection.                         .sup.b Samples taken at necropsy were serially diluted on brucella agar;      = equal to or greater than 1 CFU; 0 = no colonies.                            .sup.c SPLVs containing streptomycin sulfate, 10 mg/kg body weight, I.P.      .sup.d Streptomycin sulfate (aqueous), 10 mg/kg body weight, I.P.             .sup.e Controls received no treatment.                                   

                  TABLE VIII                                                      ______________________________________                                        RESULTS OF CULTURES FROM TISSUE                                               SAMPLES OF B. ABORTUS INFECTED COWS                                                                  SPLV-Entrapped                                                        Untreated                                                                             Streptomycin                                           Tissue           Control   1      2    3                                      ______________________________________                                        Adrenal gland L  0         0      0    0                                      Adrenal gland R  ++        0      0    +                                      Atlantal LN R    ++        +      0    +                                      Atlantal LN L    0         0      0    +                                      Axillary LN R    +++       0      +    0                                      Axillary LN L    ++        0      0    0                                      Bronchial LN     0         0      0    0                                      Cervix           0         0      0    0                                      Hepatic LN       ++++      0      0    0                                      Horn of Uterus L 0         0      0    +                                      Horn of Uterus R 0         0      0    0                                      Int. Illiac LN R ++        0      0    0                                      Int. Illiac LN L ++++      0      +    0                                      Kidney           0         0      0    0                                      Liver            0         0      0    0                                      Lung             0         0      0    0                                      Mammary Gland LF 0         +      +    0                                      Mammary Gland LR 0         0      0    +                                      Mammary Gland RF ++        0      0    0                                      Mammary Gland RR ++        0      0    0                                      Mandibular LN R  +++       0      0    0                                      Mandibular LN L  +++       0      0    0                                      Mediastinal LN   ++        0      +    0                                      Mesenteric LN    +++       0      0    0                                      Parotid LN L     +++       0      0    0                                      Parotid LN R     +++       0      0    0                                      Popliteal LN L   +         0      0    0                                      Popliteal LN R   +         0      0    0                                      Prefemoral LN L  +         0      0    0                                      Prefemoral LN R  0         0      0    0                                      Prescapular LN L 0         0      0    +                                      Prescapular LN R ++++      0      0    0                                      Renal LN         0         0      0    0                                      Spleen           +++       0      0    0                                      Supramammary LN L                                                                              +++       +      0    0                                      Supramammary LN R                                                                              0         0      0    0                                      Suprapharangeal LN L                                                                           +         0      0    0                                      Suprapharangeal LN R                                                                           0         0      0    0                                      Thymus           0         0      0    0                                      Vagina           +++       0      0    0                                      ______________________________________                                         0 No detectable bacteria by culture of 0.3-1 gm of tissue.                    + Less than 200 colonies/gm tissue.                                           ++ More than 300 colonies/gm.                                                 +++ More than 1,000 colonies/gm.                                              ++++ More than 100,000 colonies/gm.                                      

We claim:
 1. A method of treating infections in an animal byadministration of a therapeutically effective amount of aminoglycosidein liposome form by intramammary infusion.
 2. A method of treatingBrucella spp. infections in an animal by administration of atherapeutically effective amount of aminoglycoside in liposome form byintramammary infusion.
 3. The method of claim 2 wherein theaminoglycoside is streptomycin.
 4. The method of claim 3 wherein thestreptomycin is administered at from about 35 to about 50 mg/kg bodyweight.
 5. The method of claim 3 wherein the streptomycin isadministered in a dose regimen of about 2 to about 5 doses.
 6. Themethod of claim 3 wherein the liposomes comprise at least about equalamounts of lipid and streptomycin sulfate (w/w).
 7. The method of claim2 further comprising administration of a therapeutically effectiveamount of a tetracycline.
 8. The method of claim 7 wherein thetetracycline is oxytetracycline.
 9. The method of claim 8 wherein theoxytetracycline is administered at from about 10 to about 30 mg/kg bodyweight.
 10. The method of claim 2 wherein the liposomes are stableplurilamellar vesicles.
 11. The method of claim 10 wherein the liposomescomprise phosphatidylcholine.
 12. A method of administering atherapeutic agent to a proximal mammary lymph node of an animalcomprising intramammary infusion of said therapeutic agent in liposomeform.
 13. The method of claim 12 wherein the therapeutic agent is ananti-infective agent.
 14. The method of claim 13 wherein theanti-infective agent is an imidazole.
 15. The method of claim 13 whereinthe anti-infective agent is a β-lactam.
 16. The method of claim 12wherein the therapeutic agent is an antineoplastic agent.
 17. The methodof claim 16 wherein the antineoplastic agent is doxorubicin.
 18. Themethod of claim 16 wherein the antineoplastic agent is cisplatin. 19.The method of claim 16 wherein the antineoplastic agent is5-fluorouracil
 20. The method of claim 12 wherein the liposomes arestable plurilamellar vesicles.
 21. The method of claim 12 wherein theliposomes comprise phosphatidylcholine.